Yi Sheng Chang

Comparison of dyes for cataract surgery. Part 1 Cytotoxicity to corneal endothelial cells in a rabbit model

Purpose: To investigate the corneal endothelial cytotoxicity of dyes for capsule staining in cataract surgery. Setting: Department of Ophthalmology and Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Methods: Cultured corneal endothelial cells of New Zealand white rabbits were exposed for 1 minute to 1 of the following dyes (various concentrations): indocyanine green (ICG), methylene blue (MB), gentian violet (GV), trypan blue (TB), and fluorescein sodium (FS). The degree of cell damage was determined by in vitro staining with TB and comparison with results in a control group. The effect of longer exposure (up to 10 minutes) to ICG 0.25% was also investigated. Structural changes in corneal endothelial cells after dye exposure were evaluated by light microscopy and transmission electron microscopy (TEM). Results: Indocyanine green 0.25%, MB 0.20%, GV 0.01%, TB 0.40%, and FS 10% did not induce significant damage to corneal endothelial cells. Significant cytotoxicity was observed with the following or higher dye concentrations: ICG 0.50%, MB 0.50%, and GV 0.10%. Exposure to ICG 0.25% for 1 to 10 minutes showed a trend toward cytotoxicity after 10 minutes. On TEM, corneal endothelial cells that had been exposed to ICG 0.50% showed remarkable organelle swelling and disruption, electron‐dense granules, and cell lysis. Conclusion: One minute of exposure to ICG 0.25%, MB 0.20%, GV 0.01%, TB 0.40%, and FS 10% appeared to be safe as determined by no cytotoxic effects on rabbit corneal endothelial cells in culture.

Cytotoxicity of lidocaine or bupivacaine on corneal endothelial cells in a rabbit model

Purpose: To investigate the corneal endothelial cytotoxicity of commercial formulations of agents used for intracameral anesthesia in cataract and other ocular surgery. Methods: Cultured corneal endothelial cells (CECs) of New Zealand White rabbits were exposed for 1 minute to balanced salt solution (control); Xylocaine (lidocaine) 1% E (with epinephrine), 2% E, 2%, or 4%; or Marcaine (bupivacaine) 0.5% or 0.5% spinal heavy. The degree of cytotoxicity was determined by in vitro staining with trypan blue and light microscopic evaluation of cell morphology. The effect of longer exposure (up to 16 minutes) to lidocaine 1% E was also investigated. Results: CECs were not significantly damaged by 1-minute exposure to lidocaine 1% E or 2% E; however, significant cytotoxicity was seen after 1-minute exposure to lidocaine 2% or 4% or bupivacaine 0.5% or 0.5% spinal heavy. Exposure to lidocaine 1% E showed a trend toward time-dependent cytotoxicity that reached significance at 16 minutes. Conclusions: One-minute exposure to lidocaine 1% E or 2% E appears to be safe for cultured rabbit CECs, although longer exposures could cause time-dependent cytotoxicity, which should be considered in planning cataract or other ocular surgery. Because bupivacaine 0.5% and 0.5% spinal heavy cause cytotoxic effects within the first minute of contact with CECs, they should be used with great caution, if at all, in the anterior chamber of human eyes.

In vitro benzyl alcohol cytotoxicity: Implications for intravitreal use of triamcinolone acetonide

The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9mg/mL) for 5, 30, 60, or 120min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2h; but BA 0.0225mg/mL did not. BA 9.0mg/mL, the concentration in commercial TA suspensions, was toxic within 5min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225-9mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged.

Triamcinolone acetonide suspension toxicity to corneal endothelial cells.

PURPOSE: To investigate the cytotoxicity of triamcinolone acetonide (TA) suspensions to corneal endothelial cells (CECs). SETTING: Department of Ophthalmology, Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. METHODS: New Zealand white rabbit CECs were exposed for 1 minute to balanced salt solution (BSS); commercial TA suspension (cTA); vehicle‐removed TA (–vTA); pure vehicle (V); 1/10 dilutions of cTA, –vTA, or V in BSS; or benzyl alcohol (BA) (cTA preservative) 9 mg/mL. Corneal endothelial cell toxicity was assessed by light microscopy (trypan blue staining) and transmission electron microscopy. The effects of 3‐, 10‐, or 30‐minute exposures to 1/10 cTA, 1/10 –vTA, or V were also investigated. RESULTS: One‐minute exposures to –vTA or 1/10 –vTA did not damage CECs; however, cTA, V, or 1/10 dilutions of cTA or V caused damage and cells exposed to BA showed severe ultrastructural damage/lysis. A 30‐minute exposure to 1/10 –vTA did not cause significant cell damage, whereas 3‐ to 30‐minute exposures to 1/10 cTA or V showed significant time‐dependent cytotoxicity. CONCLUSIONS: Commercial TA suspension was cytotoxic to cultured rabbit CECs because of the preservative, BA, in the vehicle. Because 1/10 –vTA appeared to be safe for up to 30 minutes of exposure, use of 1/10 dilutions of vehicle‐removed TA is suggested to help surgeons visualize prolapsed vitreous during anterior vitrectomy in complicated cataract surgeries.

Comparison of dyes for cataract surgery - Part 2: Efficacy of capsule staining in a rabbit model

Purpose: To investigate the efficacy of various dyes for anterior capsule staining to facilitate capsulorhexis during cataract surgery. Setting: Department of Ophthalmology and Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Methods: Various concentrations of indocyanine green (ICG), methylene blue (MB), gentian violet (GV), trypan blue (TB), and fluorescein sodium (FS) were used to stain rabbit lenses in vitro. After 1 minute of exposure, the staining of each lens was evaluated using a semiquantitative scoring system in which 0 represented no staining/no contrast between the capsule and cortex and 4 represented excellent staining/remarkable contrast between the stained capsule and unstained cortex. The lowest concentration of dye with a score of 4 was considered the lowest effective concentration for capsulorhexis. Results: Based on our scoring system, the lowest effective concentrations for capsulorhexis were ICG 0.25%, MB 0.10%, GV 0.01%, TB 0.10%, and FS 1.25%. The lowest effective concentrations of the 5 dyes provided comparable contrast. Increased concentrations of dye decreased contrast between the capsule and the cortex and were considered less effective for capsulorhexis. Conclusion: Any of the following concentrations of dyes can be used for optimal enhancement of anterior capsule visibility for capsulorhexis: ICG 0.25%, MB 0.10%, GV 0.01%, TB 0.10%, and FS 1.25%.

Subconjunctival mitomycin C before pterygium excision: an ultrastructural study.

Purpose: Subconjunctival injection of mitomycin C (MMC) before pterygium excision is a new adjunctive therapy to decrease pterygium recurrence. This study aimed to investigate the ultrastructural changes in pterygium after subconjunctival injection of MMC. Methods: Four patients underwent subconjunctival injection of 0.1 mL of 0.15 mg/mL MMC 1 month before pterygium excision, and 2 patients served as controls without preoperative MMC injection. The excised specimens of pterygium were examined under transmission electron microscopy. Results: Epithelial cells of the treated pterygium remained unchanged. However, stromal fibroblasts were decreased in number, were oval rather than spindle-shaped, and had shrunken cytoplasmic processes; some were degenerating or apoptotic. Collagen and elastic fibers were decreased in density, disorganized, and degenerated. Capillary endothelial cells were thickened and swollen, with narrow or obliterated lumens. Axonal swelling and demyelination were observed. Conclusions: Subconjunctival injection of MMC inhibits fibrovascular activity in the pterygial stroma, leading to degeneration of the extracellular matrix and nerve axons. These ultrastructural changes are consistent with the clinical observation of reduced vascularity in the pterygium after MMC injection and verify the effectiveness of subconjunctival MMC injection 1 month before pterygium excision in decreasing the risk of pterygium recurrence.

Mechanisms Underlying Benzyl Alcohol Cytotoxicity (Triamcinolone Acetonide Preservative) in Human Retinal Pigment Epithelial Cells

PURPOSEnBenzyl alcohol (BA) is the preservative in triamcinolone acetonide (TA) suspensions, which are used in treating vitreoretinal diseases and during surgery. This paper investigates the molecular mechanisms and signaling pathways underlying BA toxicity in human retinal pigment epithelial (RPE) cells.nnnMETHODSnCultured human RPE cells from the ARPE-19 cell line were exposed to culture medium alone (control) or with BA (0.0225, 0.225, 0.9, 3, or 9 mg/mL) for up to 6 hours. BA toxicity was assessed by TUNEL assay, propidium iodide/annexin V-FITC staining and flow cytometry, caspase activation assay, caspase and apoptosis inhibition assays, mitochondrial transmembrane potential by rhodamine staining and flow cytometry, reactive oxygen species by chemiluminescence, and apoptosis-inducing factor staining.nnnRESULTSnBA caused RPE cell death not only by necrosis but also by apoptosis, evidenced by exposure to 9 mg/mL BA for 6 hours leading to 19.0% early apoptotic cells and 64.2% apoptotic necrotic cells. Apoptotic signaling involved the immediate production of reactive oxygen species, activation of caspase-8, impairment of the mitochondrial transmembrane potential, and further activation of caspase-9 and -3. In addition, BA induced translocation of apoptosis-inducing factor into the nucleus, indicating caspase-independent apoptosis.nnnCONCLUSIONSnBA leads to necrosis of RPE cells and triggers mitochondrial apoptosis through both caspase-dependent and - independent pathways. Extreme caution is suggested in the intraocular use of TA suspensions and meticulous evaluation before adoption of BA as a preservative in the future development of ophthalmic formulations.